In this study, we demonstrate how urban-dwelling bats can be used to reconstruct Urban Heat Islands (UHI). We term this approach biologically-assisted sampling (BAS). We used Egyptian fruit bats to map the spatial air temperature (Tair) profile. To demonstrate the feasibility of using biologically-assisted sampled data set, we run mixed effects and Geographically Weighted Regression (GWR) models to estimate the impact of urban environment on Tair distribution. Our results suggest that vegetation is a very important mitigating factor in Tair. In the winter, we found an average Tair difference of 2–5 ◦C between densely urban and nearby vegetative/open areas. A distinct UHI spot was identified in the winter, centered on the Ayalon highway. These differences were lower during the summer night, probably due to a pronounced cooling sea breeze effect along the coastline. Our preliminary results also indicate that BAS sampling provide a 3D view of the UHI phenomenon: the change in Tair above the dense urban area was smaller than above the vegetative area. Since the differences in Tair between densely urban and open/green areas are the largest during the night hours, bats can serve as efficient agents to monitor UHI effects, despite the method limitations.